Nucleic acid sequencing by Raman monitoring of molecular deconstruction

ABSTRACT

The disclosed methods, apparatus and compositions are of use for nucleic acid sequencing. More particularly, the methods and apparatus concern sequencing single molecules of single stranded DNA or RNA by exposing the molecule to exonuclease activity, removing free nucleotides one at a time from one end of the nucleic acid, and identifying the released nucleotides by Raman spectroscopy or FRET.

FIELD OF THE INVENTION

The present methods, compositions and apparatus relate to the fields ofmolecular biology and genomics. More particularly, the methods,compositions and apparatus concern nucleic acid sequencing.

BACKGROUND

The advent of the human genome project required that improved methodsfor sequencing nucleic acids, such as DNA (deoxyribonucleic acid) andRNA (ribonucleic acid), be developed. Genetic information is stored inthe form of very long molecules of DNA organized into chromosomes. Thetwenty-three pairs of chromosomes in the human genome containapproximately three billion bases of DNA sequence. This DNA sequenceinformation determines multiple characteristics of each individual, suchas height, eye color and ethnicity. Many common diseases, such ascancer, cystic fibrosis, sickle cell anemia and muscular dystrophy arebased at least in part on variations in DNA sequence.

Determination of the entire sequence of the human genome has provided afoundation for identifying the genetic basis of such diseases. However,a great deal of work remains to be done to identify the geneticvariations associated with each disease. That would require DNAsequencing of portions of chromosomes in individuals or familiesexhibiting each such disease, in order to identify specific changes inDNA sequence that promote the disease. RNA, an intermediary moleculerequired for processing of genetic information, can also be sequenced insome cases to identify the genetic bases of various diseases.

Existing methods for nucleic acid sequencing, based on detection offluorescently labeled nucleic acids that have been separated by size,are limited by the length of the nucleic acid that can be sequenced.Typically, only 500 to 1,000 bases of nucleic acid sequence can bedetermined at one time. This is much shorter than the length of thefunctional unit of DNA, referred to as a gene, which can be tens or evenhundreds of thousands of bases in length. Using current methods,determination of a complete gene sequence requires that many copies ofthe gene be produced, cut into overlapping fragments and sequenced,after which the overlapping DNA sequences may be assembled into thecomplete gene. This process is laborious, expensive, inefficient andtime-consuming.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the disclosedembodiments. The embodiments may be better understood by reference toone or more of these drawings in combination with the detaileddescription of specific embodiments presented herein.

FIG. 1 illustrates an exemplary apparatus (not to scale) and method forDNA sequencing in which the released nucleotides are spatially separatedfrom the nucleic acid molecule to be sequenced.

FIG. 2 illustrates an exemplary apparatus (not to scale) and method forDNA sequencing in which the released nucleotides are not spatiallyseparated from the nucleic acid molecule. The detector quantifies thenucleotides present in solution.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosed methods, compositions and apparatus are of use for therapid, automated sequencing of nucleic acids. In particular embodiments,the methods, compositions and apparatus are suitable for obtaining thesequences of very long nucleic acid molecules of greater than 1,000,greater than 2,000, greater than 5,000, greater than 10,000, greaterthan 20,000, greater than 50,000, greater than 100,000 or even morebases in length. In various embodiments, such sequence information maybe obtained during the course of a single sequencing run, using onemolecule of nucleic acid 13, 102. In other embodiments, multiple copiesof the nucleic acid molecule 13, 102 may be sequenced in parallel orsequentially to confirm the nucleic acid sequence or to obtain completesequence data. In alternative embodiments, both the nucleic acidmolecule 13, 102 and its complementary strand may be sequenced toconfirm the accuracy of the sequence information. Advantages over priormethods of nucleic acid sequencing include the ability to read longnucleic acid sequences in a single sequencing run, greater speed ofobtaining sequence data, decreased cost of sequencing and greaterefficiency in terms of the amount of operator time required per unit ofsequence data generated.

In certain embodiments, the nucleic acid 13, 102 to be sequenced is DNA,although it is contemplated that other nucleic acids 13, 102 comprisingRNA or synthetic nucleotide analogs could be sequenced as well. Thefollowing detailed description contains numerous specific details inorder to provide a more thorough understanding of the disclosedembodiments. However, it will be apparent to those skilled in the artthat the embodiments may be practiced without these specific details. Inother instances, devices, methods, procedures, and individual componentsthat are well known in the art have not been described in detail herein.

In some embodiments, disclosed in FIG. 1 and FIG. 2, the methods involvesequencing of individual single-stranded nucleic acid molecules 13, 102that are attached to an immobilization surface 14, 103 in a reactionchamber 11, 101 and disassembled in a deconstruction reaction. In suchembodiments, the reaction chamber 11, 101 contains one or moredeconstruction reagents 15, 106 that sequentially remove one nucleotide16, 104 at a time from the unattached end 17, 105 of the nucleic acidmolecule 13, 102. Non-limiting examples of such deconstruction reagents15, 106 include any exonuclease known in the art. In some embodiments,the nucleotides 16, 104 are identified by Raman spectroscopy as they arereleased into solution.

Certain embodiments are illustrated in FIG. 1. FIG. 1 shows an apparatus10 for nucleic acid sequencing comprising a reaction chamber 11 attachedto a flow path 12. The reaction chamber 11 contains a nucleic acidmolecule 13 attached to an immobilization surface 14 along with adeconstruction reagent 15, such as an exonuclease. The exonuclease 15catalyzes the sequential release of individual nucleotides 16 from thefree end 17 of the nucleic acid molecule 13. As the individualnucleotides 16 are released by the deconstruction reaction and entersolution, they move down the flow path 12 past a detection unit 18. Thedetection unit 18 comprises an excitation source 19, such as a laser,that emits an excitatory beam 20. The excitatory beam 20 interacts withthe released nucleotides 16 so that electrons are excited to a higherenergy state. The Raman emission spectrum that results from the returnof the electrons to a lower energy state is detected by a Ramanspectroscopic detector 21, such as a spectrometer or a monochromator.

In embodiments illustrated in FIG. 1, the released nucleotides 16 arespatially separated from the nucleic acid molecule 13 before detectionby the detection unit 18. Spatial separation acts to increase thesignal-to-noise ratio of the Raman detector 21 by isolating theindividual nucleotides 16.

FIG. 1 illustrates embodiments in which a single nucleic acid molecule13 is contained in a single reaction chamber 11. In alternativeembodiments, multiple nucleic acid molecules 13, each in a separatereaction chamber 11, may be sequenced simultaneously. In such cases, thenucleic acid molecule 13 in each reaction chamber 11 may be identical ormay be different. In other alternative embodiments, two or more nucleicacid molecules 13 may be present in a single reaction chamber 11. Insuch embodiments, the nucleic acid molecules 13 will be identical insequence. Where more than one nucleic acid molecule 13 is present in thereaction chamber 11, the Raman emission signals will represent anaverage of the nucleotides 16 released simultaneously from all nucleicacid molecules 13 in the reaction chamber 11. The skilled artisan willbe able to correct the signal obtained at any given time fordeconstruction reactions that either lag behind or precede the majorityof reactions occurring in the reaction chamber 11, using known dataanalysis techniques. In certain embodiments, the skilled artisan may useprocedures to synchronize the deconstruction of multiple nucleic acidmolecules 13 present in a single reaction chamber 11, as by adding abolus of deconstruction reagents 15 with rapid mixing.

In certain alternative embodiments, a tag molecule may be added to thereaction chamber 11 or to the flow path 12 upstream of the detectionunit 18. The tag molecule binds to and tags free nucleotides 16 as theyare released from the nucleic acid molecule 13. This post-releasetagging avoids problems that are encountered when the nucleotides 16 ofthe nucleic acid molecule 13 are tagged before their release intosolution. For example, the use of bulky fluorescent probe molecules mayprovide considerable steric hindrance when each nucleotide 16incorporated into a nucleic acid molecule 13 is labeled beforedeconstruction, reducing the efficiency and increasing the time requiredfor the sequencing reaction.

In embodiments involving post-release tagging of nucleotides 16, it iscontemplated that alternative methods of detection may be used, forexample fluorescence spectroscopy or luminescence spectroscopy. Manyalternative methods of detection of free nucleotides 16 in solution areknown and may be used. For such methods, the Raman spectroscopicdetection unit 18 may be replaced with a detection unit 18 designed todetect fluorescence, luminescence or other types of signals.

The tag molecules have unique and highly visible optical signatures thatcan be distinguished for each of the common nucleotides 16. In certainembodiments, the tag may serve to increase the strength of the Ramanemission signal or to otherwise enhance the sensitivity or specificityof the Raman detector 21 for nucleotides 16. Non-limiting examples oftag molecules that could be used for embodiments involving Ramanspectroscopy include TRIT (tetramethyl rhodamine isothiol), NBD(7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid,terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blueviolet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine andaminoacridine. Other tag moieties that may be of use for particularembodiments include cyanide, thiol, chlorine, bromine, methyl,phosphorus and sulfur. In certain embodiments, carbon nanotubes may beof use as Raman tags. The use of tags in Raman spectroscopy is known inthe art (e.g., U.S. Pat. Nos. 5,306,403 and 6,174,677). The skilledartisan will realize that Raman tags should generate distinguishableRaman spectra when bound to different nucleotides 16, or differentlabels should be designed to bind only one type of nucleotide 16.

In certain embodiments, the nucleic acid molecule 13 is fixed in place,as by attachment to an immobilization surface 14, and immersed in amicrofluidic flow down a flow path 12 that transports the releasednucleotides 16 away from the nucleic acid molecule 13 and past adetection unit 18. In non-limiting examples, the microfluidic flow mayresult from a bulk flow of solvent past the nucleic acid molecule 13 anddown a flow path 12, for example, a microcapillary tube or an etchedchannel in a silicon, glass or other chip. In alternative embodiments,the bulk medium moves only slowly or not at all, but charged specieswithin the solution (such as negatively charged nucleotides 16) movedown a flow path 12 comprising a channel or tube in response to anexternally applied electrical field.

In other alternative embodiments, the nucleic acid molecule 13 may bemoved by moving the immobilization surface 14 to which it is attachedaway from the released nucleotides 16. The released nucleotides 16 maybe scanned and identified by a moving detection unit 18 that follows thenucleic acid molecule 13.

In the embodiments discussed above, the detection unit 18 must becapable of distinguishing between the common nucleotides 16 releasedfrom the nucleic acid molecule 13. At a minimum, the detection unit 18must be able to distinguish between nucleotides 16 containing adenosine(A), guanosine (G), cytosine (C) and thymidine (T) for sequencing DNAmolecules 13. If RNA 13 is being sequenced, the detection unit 18 mustbe able to distinguish between nucleotides 16 containing A, G, C anduridine (U). With a single nucleic acid molecule 13 per reaction chamber11, it is not necessary that the detection unit 18 be capable ofquantifying the amounts of each nucleotide 16 in solution, since thenucleotides 16 move past the detection unit 18 one at a time.

In other embodiments, illustrated in FIG. 2, the detection unit 107 issensitive enough to quantify the number of free nucleotides 104 presentin solution. Thus, separation of released nucleotides 104 from thenucleic acid molecule 102 is not required. As illustrated in FIG. 2, theapparatus 100 comprises a reaction chamber 101 containing a nucleic acidmolecule 102 attached at one end to an immobilization surface 103. Freenucleotides 104 are sequentially removed from the unattached end 105 ofthe nucleic acid molecule 102 by the action of a deconstruction reagent106, such as an exonuclease.

As shown in FIG. 2, in these embodiments the free nucleotides 104 arenot spatially segregated from the nucleic acid molecule 102. Thedetection unit 107, comprising an excitation source 108 emitting anexcitation beam 110 and a detector 109, analyzes the reaction chamber101 containing both nucleic acid molecule 102 and free nucleotides 104.Because the detector 109 can quantify the amount of each nucleotide 104in solution, the nucleic acid 102 sequence can be determined by thetemporal sequence of release of nucleotides 104 into solution. Because agreater volume is being scanned, it may be necessary to employ a moreintense excitation beam 110 covering a broader area.

In these embodiments, as each successive nucleotide 104 is released fromthe unattached end 105 of the nucleic acid molecule 102, the nucleotide104 is identified by the increase in signal for that nucleotide 104 insolution. The Raman detector 109 can separately quantify the number ofmolecules of each nucleotide 104—adenosine monophosphate (AMP),guanosine monophosphate (GMP), cytosine monophosphate (CMP) and uridinemonophosphate (UMP) or thymidine monophosphate (TMP) in solution andseparate those signals from the baseline Raman signals produced by thenucleic acid molecule 102.

The skilled artisan will realize that analysis of DNA 13, 102 willresult in the release of deoxyribonucleosides or deoxyribonucleotides16, 104 (including thymidine), while analysis of RNA 13, 102 will resultin the release of ribonucleosides or ribonucleotides 16, 104 (includinguridine). Although nucleoside monophosphates 16, 104 will generally bethe form released by exonuclease 15, 106 activity, the embodiments arenot limited to detection of any particular form of free nucleotide ornucleoside 16, 104 but encompass any monomer 16, 104 that may bereleased from a nucleic acid 13, 102 by the activity of a deconstructionreagent 15, 106.

In further embodiments, some combination of the methods and apparatus10, 100 shown in FIG. 1 and FIG. 2 may be used. The method of FIG. 1 canuse a less sensitive detector 21, but requires more complicatedmolecular transport procedures. The method of FIG. 2 has simplifiedmolecular transport but requires a more sensitive detector 109. Avariety of intermediate approaches may be used. For example, amicrofluidics flow down a flow path 12 may be used to remove nucleotides16 that have already been detected from the area illuminated by theexcitation beam 20, while the quantification capability of the Ramandetector 21 allows detection to occur in the absence of a specifictemporal or spatial separation between the released nucleotides 16.

In certain embodiments, data from a detector 21, 109, such as aspectrometer or monochromator array, may flow to an informationprocessing system that maintains a database associating specific Ramansignatures with specific nucleotides 16, 104. The information processingsystem records the signatures detected by the detector 21, 109,correlates those signatures with the signatures of known nucleotides 16,104 in the database, and maintains a record of nucleotide 16, 104appearance that indicates the sequence of the nucleic acid molecule 13,102. The information processing system may also perform standardprocedures such as subtraction of background signals and “base-calling”determination when overlapping temporal or spatial signals are detectedfrom more than one nucleotide 16, 104.

In certain embodiments, the nucleic acid molecule 13, 102 may beattached to a surface 14, 103 such as functionalized glass, silicon,PDMS (polydimethlyl siloxane), silver or other metal coated surfaces,quartz, plastic, PTFE (polytetrafluoroethylene), PVP (polyvinylpyrrolidone), polystyrene, polypropylene, polyacrylamide, latex, nylon,nitrocellulose, a glass bead, a magnetic bead, or any other materialknown in the art that is capable of having functional groups such asamino, carboxyl, thiol, hydroxyl or Diels-Alder reactants incorporatedon its surface 14, 103.

In some embodiments, functional groups may be covalently attached tocross-linking agents so that binding interactions between nucleic acidmolecule 13, 102 and deconstruction reagent 15, 106 may occur withoutsteric hindrance. Typical cross-linking groups include ethylene glycololigomers and diamines. Attachment may be by either covalent ornon-covalent binding. Various methods of attaching nucleic acidmolecules 13, 102 to surfaces 14, 103 are known in the art and may beemployed.

Definitions

As used herein, “a” or “an” may mean one or more than one of an item.

“Nucleic acid” 13, 102 encompasses DNA, RNA, single-stranded,double-stranded or triple stranded and any chemical modificationsthereof, although single-stranded nucleic acids 13, 102 are preferred.Virtually any modification of the nucleic acid 13, 102 is contemplated.As used herein, a single stranded nucleic acid 13, 102 may be denoted bythe prefix “ss”, a double stranded nucleic acid 13, 102 by the prefix“ds”, and a triple stranded nucleic acid 13, 102 by the prefix “ts.”

A “nucleic acid” 13, 102 may be of almost any length, from 10, 20, 30,40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500,600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 30,000, 40,000,50,000, 75,000, 100,000, 150,000, 200,000, 500,000, 1,000,000,1,500,000, 2,000,000, 5,000,000 or even more bases in length, up to afull-length chromosomal DNA molecule 13, 102.

A “nucleoside” 16, 104 is a molecule comprising a base (A, C, G, T or U)covalently attached to a pentose sugar such as deoxyribose, ribose orderivatives or analogs of pentose sugars.

A “nucleotide” 16, 104 refers to a nucleoside further comprising atleast one phosphate group covalently attached to the pentose sugar. Insome embodiments, the nucleotides 16, 104 are ribonucleosidemonophosphates 16, 104 or deoxyribonucleoside monophosphates 16, 104although in certain embodiments it is anticipated that nucleosidediphosphates or triphosphates 16, 104 could be produced. In otherembodiments, nucleosides 16, 104 may be released from the nucleic acidmolecule 13, 102 and detected as discussed below. It is contemplatedthat various substitutions or modifications may be made in the structureof the nucleotides 16, 104, so long as they are still capable of beingreleased from the nucleic acid 13, 102 by a deconstruction reagent 15,106. For example, in certain embodiments the ribose or deoxyribosemoiety may be substituted with another pentose sugar or a pentose sugaranalog. In other embodiments, the phosphate groups may be substituted byvarious analogs.

Nucleic Acids

Nucleic acid molecules 13, 102 to be sequenced may be prepared by anytechnique known in the art. In certain embodiments, the nucleic acids13, 102 are naturally occurring DNA or RNA molecules. Virtually anynaturally occurring nucleic acid 13, 102 may be prepared and sequencedby the disclosed methods including, without limit, chromosomal,mitochondrial and chloroplast DNA and ribosomal, transfer, heterogeneousnuclear and messenger RNA.

Methods for preparing and isolating various forms of cellular nucleicacids 13, 102 are known. (See, e.g., Guide to Molecular CloningTechniques, eds. Berger and Kimmel, Academic Press, New York, N.Y.,1987; Molecular Cloning: A Laboratory Manual, 2nd Ed., eds. Sambrook,Fritsch and Maniatis, Cold Spring Harbor Press, Cold Spring Harbor,N.Y., 1989). Generally, cells, tissues or other source materialcontaining nucleic acids 13, 102 to be sequenced are first homogenized,for example by freezing in liquid nitrogen followed by grinding in amortar and pestle. Certain tissues may be homogenized using a Waringblender, Virtis homogenizer, Dounce homogenizer or other homogenizer.Crude homogenates may be extracted with detergents, such as sodiumdodecyl sulphate (SDS), Triton X-100, CHAPS(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate),octylglucoside or other detergents known in the art. Alternatively or inaddition, extraction may use chaotropic agents such as guanidiniumisothiocyanate, or organic solvents such as phenol. In some embodiments,protease treatment, for example with proteinase K, may be used todegrade cell proteins. Particulate contaminants may be removed bycentrifugation or ultracentrifugation (for example, 10 to 30 min atabout 5,000 to 10,000×g, or 30 to 60 min at about 50,000 to 100,000×g).Dialysis against aqueous buffer of low ionic strength may be of use toremove salts or other soluble contaminants. Nucleic acids 13, 102 may beprecipitated by addition of ethanol at −20° C., or by addition of sodiumacetate (pH 6.5, about 0.3 M) and 0.8 volumes of 2-propanol.Precipitated nucleic acids 13, 102 may be collected by centrifugationor, for chromosomal DNA 13, 102, by spooling the precipitated DNA 13,102 on a glass pipet or other probe.

The skilled artisan will realize that the procedures listed above areexemplary only and that many variations may be used, depending on theparticular type of nucleic acid 13, 102 to be sequenced. For example,mitochondrial DNA 13, 102 is often prepared by cesium chloride densitygradient centrifugation, using step gradients, while mRNA 13, 102 isoften prepared using preparative columns from commercial sources, suchas Promega (Madison, Wis.) or Clontech (Palo Alto, Calif.). Suchvariations are known in the art.

The skilled artisan will realize that depending on the type of nucleicacid 13, 102 to be prepared, various nuclease inhibitors may be used.For example, RNase contamination in bulk solutions may be eliminated bytreatment with diethyl pyrocarbonate (DEPC). Commercially availablenuclease inhibitors may be obtained from standard sources such asPromega (Madison, Wis.) or BRL (Gaithersburg, Md.). Purified nucleicacid 13, 102 may be dissolved in aqueous buffer, such as TE (Tris-EDTA)(ethylene diamine tetraacetic acid) and stored at −20° C. or in liquidnitrogen prior to use.

In cases where single stranded DNA (ssDNA) 13, 102 is to be sequenced, assDNA 13, 102 may be prepared from double stranded DNA (dsDNA) bystandard methods. Most simply, dsDNA may be heated above its annealingtemperature, at which point it spontaneously separates into ssDNA 13,102. Representative conditions might involve heating at 92 to 95° C. for5 min or longer. Formulas for determining conditions to separate dsDNA,based for example on GC content and the length of the molecule, areknown in the art. Alternatively, single-stranded DNA 13, 102 may beprepared from double-stranded DNA by standard amplification techniquesknown in the art, using a primer that only binds to one strand ofdouble-stranded DNA. Other methods of preparing single-stranded DNA 13,102 are known in the art, for example by inserting the double-strandednucleic acid to be sequenced into the replicative form of a phage likeM13, and allowing the phage to produce single-stranded copies of thenucleic acid 13, 102.

Although certain embodiments concern preparation of naturally occurringnucleic acids 13, 102, virtually any type of nucleic acid 13, 102 thatcan serve as a substrate for an exonuclease or other deconstructionreagent 15, 106 could potentially be sequenced. For example, nucleicacids 13, 102 prepared by various amplification techniques, such aspolymerase chain reaction (PCRm) amplification, could be sequenced. (SeeU.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159.) Nucleic acids 13,102 to be sequenced may alternatively be cloned in standard vectors,such as plasmids, cosmids, BACs (bacterial artificial chromosomes) orYACs (yeast artificial chromosomes). (See, e.g., Berger and Gmmel, 1987;Sambrook et al., 1989.) Nucleic acid inserts 13, 102 may be isolatedfrom vector DNA, for example, by excision with appropriate restrictionendonucleases, followed by agarose gel electrophoresis and ethidiumbromide staining. Selected size-fractionated nucleic acids 13, 102 maybe removed from gels, for example by the use of low melting pointagarose or by electroelution from gel slices. Methods for isolation ofinsert nucleic acids 13, 102 are known in the art.

Isolation of Single Nucleic Acid Molecules

In certain embodiments, the nucleic acid molecule 13, 102 to besequenced is a single molecule of ssDNA or ssFWA. A variety of methodsfor selection and manipulation of single ssDNA or ssRNA molecules 13,102 may be used, for example, hydrodynamic focusing, micro-manipulatorcoupling, optical trapping, or a combination of these and similarmethods. (See, e.g., Goodwin et al., 1996, Acc. Chem. Res. 29:607-619;U.S. Pat. Nos. 4,962,037; 5,405,747; 5,776,674; 6,136,543; 6,225,068.)

In certain embodiments, microfluidics or nanofluidics may be used tosort and isolate nucleic acid molecules 13, 102. Hydrodynamics may beused to manipulate the movement of nucleic acids 13, 102 into amicrochannel, microcapillary, or a micropore. In one embodiment,hydrodynamic forces may be used to move nucleic acid molecules 13, 102across a comb structure to separate single nucleic acid molecules 13,102. Once the nucleic acid molecules 13, 102 have been separated,hydrodynamic focusing may be used to position the molecules 13, 102within the reaction chamber 11, 101. A thermal or electric potential,pressure or vacuum can also be used to provide a motive force formanipulation of nucleic acids 13, 102. In exemplary embodiments,manipulation of nucleic acids 13, 102 for sequencing may involve the useof a channel block design incorporating microfabricated channels and anintegrated gel material, as disclosed in U.S. Pat. Nos. 5,867,266 and6,2 14,246.

In another embodiment, a sample containing the nucleic acid molecule 13,102 may be diluted prior to coupling to an immobilization surface 14,103. In exemplary embodiments, the immobilization surface 14, 103 may bein the form of magnetic or non-magnetic beads or other discretestructural units. At an appropriate dilution, each bead 14, 103 willhave a statistical probability of binding zero or one nucleic acidmolecule 13, 102. Beads 14, 103 with one attached nucleic acid molecule13, 102 may be identified using, for example, fluorescent dyes and flowcytometer sorting or magnetic sorting. Depending on the relative sizesand uniformity of the beads 14, 103 and the nucleic acids 13, 102, itmay be possible to use a magnetic filter and mass separation to separatebeads 14, 103 containing a single bound nucleic acid molecule 13, 102.In other embodiments, multiple nucleic acids 13, 102 attached to asingle bead or other immobilization surface 14, 103 may be sequenced.

In alternative embodiments, a coated fiber tip 14, 103 may be used togenerate single molecule nucleic acids 13, 102 for sequencing (e.g.,U.S. Pat. No. 6,225,068). In other alternative embodiments, theimmobilization surfaces 14, 103 may be prepared to contain a singlemolecule of avidin or other cross-linking agent. Such a surface 14, 103could attach a single biotinylated nucleic acid molecule 13, 102 to besequenced. This embodiment is not limited to the avidin-biotin bindingsystem, but may be adapted to any coupling system known in the art.

In other alternative embodiments, an optical trap may be used formanipulation of single molecule nucleic acid molecules 13, 102 forsequencing. (E.g., U.S. Pat. No. 5,776,674). Exemplary optical trappingsystems are commercially available from Cell Robotics, Inc.(Albuquerque, N. Mex.), S+L GmbH (Heidelberg, Germany) and P.A.L.M. Gmbh(Wolfratshausen, Germany).

Methods of Immobilization

In various embodiments, the nucleic acid molecules 13, 102 to besequenced may be attached to a solid surface 14, 103 (or immobilized).Immobilization of nucleic acid molecules 13, 102 may be achieved by avariety of methods involving either non-covalent or covalent attachmentbetween the nucleic acid molecule 13, 102 and the surface 14, 103. In anexemplary embodiment, immobilization may be achieved by coating asurface 14, 103 with streptavidin or avidin and the subsequentattachment of a biotinylated nucleic acid 13, 102 (Holmstrom et al.,Anal. Biochem. 209:278-283, 1993). Immobilization may also occur bycoating a silicon, glass or other surface 14, 103 with poly-L-Lys(lysine) or poly L-Lys, Phe (phenylalanine), followed by covalentattachment of either amino- or sulfhydryl-modified nucleic acids 13, 102using bifunctional crosslinking reagents (Running et al., BioTechnigues8:276-277, 1990; Newton et al., Nucleic Acids Res. 21:1155-62, 1993).Amine residues may be introduced onto a surface 14, 103 through the useof aminosilane for cross-linking.

Immobilization may take place by direct covalent attachment of5′-phosphorylated nucleic acids 13, 102 to chemically modified surfaces14, 103 (Rasmussen et al., Anal. Biochem. 198:138-142, 1991). Thecovalent bond between the nucleic acid 13, 102 and the surface 14, 103is formed by condensation with a water-soluble carbodiimide. This methodfacilitates a predominantly 5′-attachment of the nucleic acids 13, 102via their 5′-phosphates.

DNA 13, 102 is commonly bound to glass by first silanizing the glasssurface 14, 103, then activating with carbodiimide or glutaraldehyde.Alternative procedures may use reagents such as3-glycidoxypropyltrimethoxysilane (GOP) or aminopropyltrimethoxysilane(APTS) with DNA 13, 102 linked via amino linkers incorporated either atthe 3′ or 5′ end of the molecule. DNA 13, 102 may be bound directly tomembrane surfaces 14, 103 using ultraviolet radiation. Othernon-limiting examples of immobilization techniques for nucleic acids 13,102 are disclosed in U.S. Pat. Nos. 5,610,287, 5,776,674 and 6,225,068.

The type of surface 14, 103 to be used for immobilization of the nucleicacid 13, 102 is not limiting. In various embodiments, the immobilizationsurface 14, 103 may be magnetic beads, non-magnetic beads, a planarsurface, a pointed surface, or any other conformation of solid surface14, 103 comprising almost any material, so long as the material issufficiently durable and inert to allow the nucleic acid 13, 102sequencing reaction to occur. Non-limiting examples of surfaces 14, 103that may be used include glass, silica, silicate, PDMS, silver or othermetal coated surfaces, nitrocellulose, nylon, activated quartz,activated glass, polyvinylidene difluoride (PVDF), polystyrene,polyacrylamide, other polymers such as poly(vinyl chloride), poly(methylmethacrylate) or poly(dimethyl siloxane), and photopolymers whichcontain photoreactive species such as nitrenes, carbenes and ketylradicals capable of forming covalent links with nucleic acid molecules13, 102 (See U.S. Pat. Nos. 5,405,766 and 5,986,076).

Bifunctional cross-linking reagents may be of use in variousembodiments, such as attaching a nucleic acid molecule 13, 102 to asurface 14, 103. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino,guanidino, indole, or carboxyl specific groups. Of these, reagentsdirected to free amino groups are popular because of their commercialavailability, ease of synthesis and the mild reaction conditions underwhich they can be applied. Exemplary methods for cross-linking moleculesare disclosed in U.S. Pat. Nos. 5,603,872 and 5,401,511. Cross-linkingreagents include glutaraldehyde (GAD), bifunctional oxirane (OXR),ethylene glycol diglycidyl ether (EGDE), and carbodiimides, such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

Deconstruction Reagent

The sequencing reaction involves binding of a deconstruction reagent 15,106 to the free end 17, 105 of the nucleic acid molecule 13, 102 andremoval of nucleotides 16, 104 one at a time. In certain embodiments thereaction may be catalyzed by an enzyme, such as an exonuclease 15, 106.The embodiments are not limited by the type of exonuclease 15, 106 thatmay be used. Non-limiting examples of exonucleases 15, 106 of potentialuse include E. coli exonuclease I, III, V or VII, Bal31 exonuclease,mung bean exonuclease, S1 nuclease, E. coli DNA polymerase I holoenzymeor Klenow fragment, RecJ, exonuclease T, T4 or T7 DNA polymerase, Taqpolymerase, exonuclease T7 gene 6, snake venom phosphodiesterase, spleenphosphodiesterase, Thermococcus litoralis DNA polymerase, Pyrococcus sp.GB-D DNA polymerase, lambda exonuclease, S. aureus micrococcal nuclease,DNase I, ribonuclease A, T1 micrococcal nuclease, or other exonucleasesknown in the art. Exonucleases 15, 106 are available from commercialsources such as New England Biolabs (Beverly, Mass.), Amersham PharmaciaBiotech (Piscataway, N.J.), Promega (Madison, Wis.), Sigma Chemicals(St. Louis, Mo.) or Boehringer Mannheim (Indianapolis, Ind.).

The skilled artisan will realize that enzymes with exonuclease 15, 106activity have various properties, for example, they can removenucleotides 16, 104 from the 5′ end, the 3′ end, or either end of thenucleic acid molecule 13, 102. They can show specificity for RNA, DNA orboth RNA and DNA 13, 102. Their activity may depend on the use of eithersingle or double-stranded nucleic acids 13, 102. They may bedifferentially affected by various characteristics of the reactionmedium, such as salt, temperature, pH, or divalent cations. These andother properties of the various exonucleases and polymerases 15, 106 areknown in the art.

The skilled artisan will realize that the rate of exonuclease 15, 106activity may be manipulated to coincide with the optimal rate ofanalysis of nucleotides 16, 104 by the detection unit 18, 107. Variousmethods are known for adjusting the rate of exonuclease 15, 106activity, including adjusting the temperature, pressure, pH, saltconcentration or divalent cation concentration in the reaction chamber11, 101. Methods of optimization of exonuclease 15, 106 activity areknown in the art.

Labels

Certain embodiments may involve incorporating a label into thenucleotides 16, 104, to facilitate their measurement by the detectionunit 18, 107. A number of different labels may be used, such as Ramantags, fluorophores, chromophores, radioisotopes, enzymatic tags,antibodies, chemiluminescent, electroluminescent, affinity labels, etc.One of skill in the art will recognize that these and other labelmoieties not mentioned herein can be used in various embodiments.

Labels for use in embodiments involving Raman spectroscopy are discussedabove. In other embodiments, the label moiety to be used may be afluorophore, such as Alexa 350, Alexa 430, AMCA(7-amino-4-methylcoumarin-3-acetic acid), BODIPY(57-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid) 630/650,BODIPY 650/665, BODIPY-FL (fluorescein), BODIPY-R6G(6-carboxyrhodamine), BODIPY-TMR (tetramethylrhodamine), BODIPY-TRX(Texas Red-X), Cascade Blue, Cy2 (cyanine), Cy3, Cy5, 6-FAM(5-carboxyfluorescein), Fluorescein, 6-JOE(2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein), Oregon Green 488,Oregon Green 500, Oregon Green 514, Pacific Blue, Rhodamine Green,Rhodamine Red, ROX (6-carboxy-X-rhodamine), TAMRA(N,N,N′,N′-tetramethyl-6-carboxyrhodamine), Tetramethylrhodamine, andTexas Red. Fluorescent or luminescent labels can be obtained fromstandard commercial sources, such as Molecular Probes (Eugene, Oreg.).

Reaction Chamber

The reaction chamber 11, 101 is designed to hold the immobilizationsurface 14, 103, nucleic acid molecule 13, 102, deconstruction reagent15, 106 and nucleotides 16, 104 in an aqueous environment. In someembodiments, the reaction chamber 11, 101 is designed to be temperaturecontrolled, for example by incorporation of Pelletier elements or othermethods known in the art. Methods of controlling temperature for lowvolume liquids are known in the art. (See, e.g., U.S. Pat. Nos.5,038,853, 5,919,622, 6,054,263 and 6,180,372.)

In certain embodiments, the reaction chamber 11, 101 and any associatedfluid channels, for example, the flow path 12 or channels to provideconnections to a waste port, to a nucleic acid 13, 102 loading port, orto a source of deconstruction reagent 15, 106 are manufactured in abatch fabrication process, as known in the fields of computer chipmanufacture or microcapillary chip manufacture. In some embodiments, thereaction chamber 11, 101 and other components of the apparatus 10, 100,such as the flow path 12, may be manufactured as a single integratedchip. Such a chip may be manufactured by methods known in the art, suchas by photolithography and etching. However, the manufacturing method isnot limiting and other methods known in the art may be used, such aslaser ablation, injection molding, casting, or imprinting techniques.Methods for manufacture of nanoelectromechanical systems may be used forcertain embodiments. (See, e.g., Craighead, Science 290: 1532-36, 2000.)Microfabricated chips are commercially available from sources such asCaliper Technologies Inc. (Mountain View, Calif.) and ACLARA BioSciencesInc. (Mountain View, Calif.).

In a non-limiting example, Borofloat glass wafers (Precision Glass &Optics, Santa Ana, Calif.) may be pre-etched for a short period inconcentrated HF (hydrofluoric acid) and cleaned before deposition of anamorphous silicon sacrificial layer in a plasma-enhanced chemical vapordeposition (PECVD) system (PEII-A, Technics West, San Jose, Calif.).Wafers may be primed with hexamethyldisilazane (∈&IDS), spin-coated withphotoresist (Shipley 1818, Marlborough, Mass.) and soft-baked. A contactmask aligner (Quintel Corp. San Jose, Calif.) may be used to expose thephotoresist layer with one or more mask designs, and the exposedphotoresist removed using a mixture of Microposit developer concentrate(Shipley) and water. Developed wafers may be hard-baked and the exposedamorphous silicon removed using CF4 (carbon tetrafluoride) plasma in aPECVD reactor. Wafers may be chemically etched with concentrated HF toproduce the reaction chamber 11, 101, flow path 12 and any channels. Theremaining photoresist may be stripped and the amorphous silicon removed.

Access holes may be drilled into the etched wafers with a diamond drillbit (Crystalite, Westerville, Ohio). A finished chip may be prepared bythermally bonding an etched and drilled plate to a flat wafer of thesame size in a programmable vacuum furnace (Centurion VPM, J. M. Ney,Yucaipa, Calif.). In certain embodiments, the chip may be prepared bybonding two etched plates to each other. Alternative exemplary methodsfor fabrication of a chip incorporating a reaction chamber 11, 101 andflow path 12 are disclosed in U.S. Pat. Nos. 5,867,266 and 6,214,246.

To facilitate detection of nucleotides 16, 104 by the detection unit 18,107 the material comprising the reaction chamber 11, 101 and/or flowpath 12 may be selected to be transparent to electromagnetic radiationat the excitation and emission frequencies used for the detection unit18, 107. Glass, silicon, and any other materials that are generallytransparent in the frequency ranges used for Raman spectroscopy,fluorescence spectroscopy, luminescence spectroscopy, or other forms ofspectroscopy may be used. In some embodiments the surfaces of thereaction chamber 11, 101 and/or flow path 12 that are opposite thedetection unit 18, 107 may be coated with silver, gold, platinum,copper, aluminum or other materials that are relatively opaque to thedetection unit 18, 107. In that position, the opaque material isavailable to enhance the Raman or other signal, for example by surfaceenhanced Raman spectroscopy, while not interfering with the function ofthe detection unit 18, 107. Alternatively, the reaction chamber 11, 101and/or flow path 12 may contain a mesh comprising silver, gold,platinum, copper or aluminum. The skilled artisan will realize that inembodiments involving a flow path 12, the nucleotides 16 will generallybe detected while they are in the flow path 12. In embodiments without aflow path 12, the nucleotides 104 will be detected in the reactionchamber 101.

In various embodiments, the reaction chamber 11, 101 may have aninternal volume of about 1 picoliter, about 2 picoliters, about 5picoliters, about 10 picoliters, about 20 picoliters, about 50picoliters, about 100 picoliters, about 250 picoliters, about 500picoliters, about 1 nanoliter, about 2 nanoliters, 5 nanoliters, about10 nanoliters, about 20 nanoliters, about 50 nanoliters, about 100nanoliters, about 250 nanoliters, about 500 nanoliters, about 1microliter, about 2 microliters, about 5 microliters, about 10microliters, about 20 microliters, about 50 microliters, about 100microliters, about 250 microliters, about 500 microliters, or about 1milliliter.

Flow Path

In certain embodiments, the free nucleotides 16 are moved down a flowpath 12 past the detection unit 18. A non-limiting example of techniquesfor transport of free nucleotides 16 includes microfluidic techniques.The flow path 12 can comprise a microcapillary (available, e.g., fromACLARA BioSciences Inc., Mountain View, Calif.) or a liquid integratedcircuit (e.g., Caliper Technologies Inc., Mountain View, Calif.). Suchmicrofluidic platforms require only nanoliter volumes of sample.

In certain embodiments, the free nucleotides 16 to be detected move downthe flow path 12 by bulk flow of solvent. In other embodiments,microcapillary electrophoresis may be used to transport free nucleotides16 down the flow path 12 and past the detection unit 18. Microcapillaryelectrophoresis generally involves the use of a thin capillary orchannel that may or may not be filled with a particular separationmedium. Electrophoresis of appropriately charged molecular species, suchas negatively charged nucleotides 16, occurs in response to an imposedelectrical field, negative on the reaction chamber 11 side of theapparatus and positive on the detection unit 18 side. Althoughelectrophoresis is often used for size separation of a mixture ofcomponents that are simultaneously added to the microcapillary, it canalso be used to transport similarly sized nucleotides 16 that aresequentially added to the flow path 12. Because the purine nucleotides(A, G) 16 are larger than the pyrimidine nucleotides (C, T, U) 16 andwould therefore migrate more slowly, the length of the flow path 12 andcorresponding transit time past the detector unit 18 should be kept to aminimum to prevent differential migration from mixing up the order ofnucleotides 16 released from the nucleic acid 13. Alternatively, theseparation medium filling the microcapillary may be selected so that themigration rates of purine and pyrimidine nucleotides 16 down the flowpath 12 are similar or identical. Methods of microcapillaryelectrophoresis have been disclosed, for example, by Woolley and Mathies(Proc. Natl. Acad. Sci. USA 91:11348-352, 1994).

Microfabrication of microfluidic devices, including microcapillaryelectrophoretic devices has been discussed in, e.g., Jacobsen et al.(Anal. Biochem, 209:278-283, 1994); Effenhauser et al. (Anal. Chem.662949-2953, 1994); Harrison et ol. (Science 261 :895-897, 1993) andU.S. Pat. No. 5,904,824. Typically, these methods comprisephotolithographic etching of micron scale channels on silica, silicon orother crystalline substrates or chips, and can be readily adapted foruse. In some embodiments, the microcapillary may be fabricated from thesame polymeric materials described for the fabrication of the reactionchamber 11, 101, using injection molding or other techniques known inthe art.

Detection Unit

Embodiments Involving Raman Spectroscopy

In some embodiments, the detection unit 18, 107 is designed to detectand quantify nucleotides 16, 104 by Raman spectroscopy. Various methodsfor detection of nucleotides 16, 104 by Raman spectroscopy are known inthe art. (See, e.g., U.S. Pat. Nos. 5,306,403; 6,002,471; 6,174,677).Variations on surface enhanced Raman spectroscopy (SERS) or surfaceenhanced resonance Raman spectroscopy (SERRS) have been disclosed. InSERS and SERRS, the sensitivity of the Raman detection is enhanced by afactor of 1o6 or more for molecules adsorbed on roughened metalsurfaces, such as silver, gold, platinum, copper or aluminum surfaces.

A non-limiting example of a Raman detection unit 18, 107 is disclosed inU.S. Pat. No. 6,002,471. In this embodiment, the excitation beam 20, 110is generated by either a Nd:YAG laser 19, 108 at 532 nm wavelength or aTi:sapphire laser 19, 108 at 365 nm wavelength. Pulsed laser beams 20,110 or continuous laser beams 20, 110 may be used. The excitation beam20, 110 passes through confocal optics and a microscope objective, andis focused onto the flow path 12 or the reaction chamber 101. The Ramanemission light from the nucleotides 16, 104 is collected by themicroscope objective and the confocal optics and is coupled to amonochromator for spectral dissociation. The confocal optics includes acombination of dichroic filters, barrier filters, confocal pinholes,lenses, and mirrors for reducing the background signal. Standard fullfield optics can be used as well as confocal optics. The Raman emissionsignal is detected by a Raman detector 21, 109. The detector 21, 109includes an avalanche photodiode interfaced with a computer for countingand digitization of the signal. In certain embodiments, a meshcomprising silver, gold, platinum, copper or aluminum may be included inthe flow path 12 or the reaction chamber 101 to provide an increasedsignal due to surface enhanced Raman or surface enhanced Ramanresonance.

Alternative embodiments of detection units 18, 107 are disclosed, forexample, in U.S. Pat. No. 5,306,403, including a Spex Model 1403double-grating spectrophotometer 21, 109 equipped with agallium-arsenide photomultiplier tube (RCA Model C31034 or BurleIndustries Model C3 103402) operated in the single-photon counting mode.The excitation source 19, 108 is a 514.5 nm line argon-ion laser 19, 108from SpectraPhysics, Model 166, and a 647.1 nm line of a krypton-ionlaser 19, 108 (Innova 70, Coherent).

Alternative excitation sources 19, 108 include a nitrogen laser 19, 108(Laser Science Inc.) at 337 nm and a helium-cadmium laser 19, 108(Liconox) at 325 nm (U.S. Pat. No. 6,174,677). The excitation beam 20,110 may be spectrally purified with a bandpass filter (Corion) and maybe focused on the flow path 12 or reaction chamber 101 using a 6×objective lens (Newport, Model L6X). The objective lens may be used toboth excite the nucleotides 16, 104 and to collect the Raman signal, byusing a holographic beam splitter (Kaiser Optical Systems, Inc., ModelHB 647-26N18) to produce a right-angle geometry for the excitation beam20, 110 and the emitted Raman signal. A holographic notch filter (KaiserOptical Systems, Inc.) may be used to reduce Rayleigh scatteredradiation. Alternative Raman detectors 21, 109 include an ISA HR-320spectrograph equipped with a red-enhanced intensified charge-coupleddevice (RE-ICCD) detection system (Princeton Instruments). Other typesof detectors 21, 109 may be used, such as charged injection devices,photodiode arrays or phototransistor arrays.

Any suitable form or configuration of Raman spectroscopy or relatedtechniques known in the art may be used for detection of nucleotides 16,104, including but not limited to normal Raman scattering, resonanceRaman scattering, surface enhanced Raman scattering, surface enhancedresonance Raman scattering, coherent anti-Stokes Raman spectroscopy(CARS), stimulated Raman scattering, inverse Raman spectroscopy,stimulated gain Raman spectroscopy, hyper-Raman scattering, molecularoptical laser examiner (MOLE) or Raman microprobe or Raman microscopy orconfocal Raman microspectrometry, three-dimensional or scanning Raman,Raman saturation spectroscopy, time resolved resonance Raman, Ramandecoupling spectroscopy or UV-Raman microscopy.

Embodiments Involving FRET

In certain alternative embodiments, the nucleotides 16, 104 areidentified and quantified using fluorescence resonance energy transfer(FRET). FRET is a spectroscopic phenomenon used to detect proximitybetween a donor molecule and an acceptor molecule. The donor andacceptor pairs are chosen such that fluorescent emission from the donoroverlaps the excitation spectrum of the acceptor. When the two moleculesare associated (at a distance of less than 100 Angstroms), theexcited-state energy of the donor is transferred non-radiatively to theacceptor and the donor emission is quenched. If the acceptor molecule isa fluorophore then its emission is enhanced. Compositions and methodsfor use of FRET with oligonucleotides are known in the art (e.g., U.S.Pat. No. 5,866,366).

Molecules that are frequently used as tags for FRET include fluorescein,5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonicacid (EDANS). Other potential FRET donor or acceptor molecules are knownin the art (See U.S. Pat. No. 5,866,336, Table 1). The skilled artisanwill be familiar with the selection of pairs of tag molecules for FRET(U.S. Pat. No. 5,866,336).

In embodiments involving FRET, the donor and acceptor molecules may becovalently or non-covalently attached to various constituents of thesequencing apparatus 10, 100. In certain embodiments, the donor oracceptor molecules may be attached to the nucleotides 16, 104, to theexonuclease 15, 106, or to the flow path 12.

In certain embodiments, the donor molecule may be attached to theexonuclease 15, 106 or to the surface of the flow path 12 and theacceptor molecules attached to the nucleotides 16, 104. In this case,each type of nucleotide 16, 104 should be attached to an acceptormolecule with a distinguishable emission spectrum, while the donormolecule should be selected to have a broad emission spectrum thatoverlaps with the excitation spectra for all four of the acceptormolecules. Multiple donor molecules may be present on the exonuclease15, 106 or flow path 12, although in other embodiments only a singledonor molecule may be present.

Upon excitation, the donor molecules will transfer their energy to theacceptor tag molecules attached to the nucleotides 16, 104, resulting inan enhanced emission signal from the acceptor molecules. Because thestrength of the signal enhancement decreases rapidly with distance, thegreatest signal enhancement will occur for nucleotides 16, 104 that arevery close to the donor molecule(s). In the case of a donor moleculeattached to the exonuclease 15, 106, at or near the catalytic site, thenucleotide 16, 104 with the strongest emission signal will be located atthe catalytic site of the exonuclease 15, 106. The donor molecule shouldbe attached close to the catalytic site, but in a position where it willnot interfere with the exonuclease activity of the deconstructionreagent 15, 106. In embodiments where donor molecules are attached tothe surface of the flow path 12 in the location where the excitationbeam 20 is focused, only nucleotides 16 within the focus of theexcitation beam 20 should give a detectable fluorescent signal. Thewavelength of the excitation beam 20, 110 may be selected to maximallyexcite the donor molecules, while only weakly exciting the acceptormolecules. As each nucleotide 16, 104 is removed from the nucleic acidmolecule 13, 102, the signal from its donor tag will be detected.

In certain embodiments, the template nucleic acid 13, 102 to besequenced may be held within the field of view of a fluorescencemicroscope by methods known in the art, for example by use of an opticaltrap (e.g., U.S. Pat. No. 6,136,543). A non-limiting example of afluorescence microscope that may be used is an inverted phase-contrastand incident-light fluorescence microscope (IMT2-RFC, Olympus Co.,Ltd.), using an oil-immersed 100 power lens(Plan.multidot.Apochromat.times.100, 1.40 NA, Olympus Co., Ltd.) Theexcitation beam 20, 110 may be emitted by a laser 19, 108, as discussedabove. Fluorescence emission may be collected through the objectivelens, using appropriate filters, and detected using any sensitivefluorescence detector 21, 109, such as a CCD device, photodiodes,photomultiplier tubes, or the equivalent.

Information Processing and Control System and Data Analysis

In certain embodiments, the nucleic acid sequencing apparatus 10, 100may comprise an information processing system. The embodiments are notlimiting for the type of information processing system used. Anexemplary information processing system may incorporate a computercomprising a bus for communicating information and a processor forprocessing information. In one embodiment, the processor is selectedfrom the Pentium® family of processors, including without limitation thePentium® II family, the Pentium® III family and the Pentium® 4 family ofprocessors available from Intel Corp. (Santa Clara, Calif.). Inalternative embodiments, the processor may be a Celeron®, an Itanium®,or a Pentium Xeon® processor (Intel Corp., Santa Clara, Calif.). Invarious other embodiments, the processor may be based on Intel®architecture, such as Intel® IA-32 or Intel® IA-64 architecture.Alternatively, other processors may be used.

The computer may further comprise a random access memory (RAM) or otherdynamic storage device, a read only memory (ROM) or other static storageand a data storage device such as a magnetic disk or optical disc andits corresponding drive. The information processing system may alsocomprise other peripheral devices known in the art, such a displaydevice (e.g., cathode ray tube or Liquid Crystal Display), analphanumeric input device (e.g., keyboard), a cursor control device(e.g., mouse, trackball, or cursor direction keys) and a communicationdevice (e.g., modem, network interface card, or interface device usedfor coupling to Ethernet, token ring, or other types of networks).

In particular embodiments, the detection unit 18, 107 may be operablycoupled to the information processing system. Data from the detectionunit 18, 107 may be processed by the processor and data stored in themain memory. Data on emission profiles for standard nucleotides 16, 104may also be stored in main memory or in ROM. The processor may comparethe emission spectra from nucleotides 16, 104 in the reaction chamber101 or the flow path 12 to identify the type of nucleotide 16, 104released from the nucleic acid molecule 13, 102. The main memory mayalso store the sequence of nucleotides 16, 104 released from the nucleicacid molecule 13, 102. The processor may analyze the data from thedetection unit 18, 107 to determine the sequence of the nucleic acid 13,102. It is appreciated that a differently equipped informationprocessing system may be used for certain implementations. Therefore,the configuration of the system may vary in different embodiments.

It should be noted that, while the processes described herein may beperformed under the control of a programmed processor, in alternativeembodiments, the processes may be fully or partially implemented by anyprogrammable or hardcoded logic, such as Field Programmable Gate Arrays(FPGAs), ‘ITL logic, or Application Specific Integrated Circuits(ASICs), for example. Additionally, the disclosed methods may beperformed by any combination of programmed general purpose computercomponents and/or custom hardware components.

Following the data gathering operation, the data will typically bereported to a data analysis operation. To facilitate the analysisoperation, the data obtained by the detection unit 18, 109 willtypically be analyzed using a digital computer such as that describedabove. Typically, the computer will be appropriately programmed forreceipt and storage of the data from the detection unit 18, 109 as wellas for analysis and reporting of the data gathered.

In certain embodiments, custom designed software packages may be used toanalyze the data obtained from the detection unit 18, 109. Inalternative embodiments, data analysis may be performed, using aninformation processing system and publicly available software packages.Non-limiting examples of available software for DNA sequence analysisinclude the PRISMm DNA Sequencing Analysis Software (Applied Biosystems,Foster City, Calif.), the Sequencher™ package (Gene Codes, Ann Arbor,Mich.), and a variety of software packages available through theNational Biotechnology Information Facility at websitewww.nbif.orgilinksll.4.1.php.

1-16. (canceled)
 17. A method of sequencing nucleic acids comprising: a.preparing a single nucleic acid molecule, one end of the nucleic acidmolecule attached to an immobilization surface within a reactionchamber; b. sequentially removing nucleotides from the unattached end ofthe nucleic acid molecule; c. separating the nucleotides from thenucleic acid molecule; and d. detecting the nucleotides by Ramanspectroscopy.
 18. The method of claim 17, wherein the nucleotides areremoved from the unattached end of the nucleic acid molecule byexonuclease activity.
 19. The method of claim 17, wherein a tag moleculeis attached to each nucleotide after the nucleotide is removed from thenucleic acid molecule.
 20. The method of claim 17, wherein thenucleotides are detected by surface enhanced Raman scattering, surfaceenhanced resonance Raman scattering, stimulated Raman scattering,inverse Raman, stimulated gain Raman spectroscopy, hyper-Ramanscattering or coherent anti-Stokes Raman scattering.
 21. A method ofsequencing nucleic acids comprising: a. preparing a single nucleic acidmolecule, one end of the nucleic acid molecule attached to animmobilization surface within a reaction chamber; b. sequentiallyremoving nucleotides from the unattached end of the nucleic acidmolecule; and c. quantifying the nucleotides in the reaction chamber byRaman spectroscopy.
 22. The method of claim 21, wherein the nucleotidesare removed from the unattached end of the nucleic acid molecule byexonuclease activity.
 23. The method of claim 21, wherein a tag moleculeis attached to each nucleotide after the nucleotide is removed from thenucleic acid molecule.
 24. The method of claim 21, wherein thenucleotides are detected by surface enhanced Raman scattering, surfaceenhanced resonance Raman scattering, stimulated Raman scattering,inverse Raman, stimulated gain Raman spectroscopy, hyper-Ramanscattering or coherent anti-Stokes Raman scattering.
 25. A method ofsequencing nucleic acids comprising: a. preparing a single nucleic acidmolecule, one end of the nucleic acid molecule attached to animmobilization surface within a reaction chamber; b. sequentiallyremoving nucleotides from the unattached end of the nucleic acidmolecule; and c. detecting the nucleotides by fluorescence resonanceenergy transfer (FRET) spectroscopy.
 26. The method of claim 25, whereinthe nucleotides are attached to acceptor molecules after they areremoved from the nucleic acid molecule.
 27. The method of claim 26,wherein the nucleotides are removed by an exonuclease.
 28. The method ofclaim 27, wherein one or more donor molecules is attached to theexonuclease.